Many diseases have early and chronic stage manifestation of biomarkers that help identify the disease. Most diseases are well characterized and if detected early no irreparable harm can be inflicted on a person. For example, Periodontitis is a chronic disease which affects at least 10% of the population. If untreated, Periodontitis can lead to teeth loss. Unfortunately, current diagnostic tests are limited in their sensitivity and specificity. It is characterized by apical migration of epithelial attachment, loss of connective tissue, alveolar bone and eventually tooth. Periodontitis progression is usually site specific, but is not consistent. Thus, it is difficult to clinically distinguish the progressing from the non-progressing inflamed sites. Moreover, the early stages of periodontal disease progression, particularly gingivitis, are often difficult to quantify because of the lack of a linear measurement tool.
In clinical practice, Periodontitis can be diagnosed by radiographic examinations. But still the best available diagnostic aid is the measurement of the depth of the tooth pocket. However, this only provides a retrospective analysis mostly when tooth attachment has already been lost. So, the current diagnostic methodologies are limited to identify the cause of disease or patients at risk. In addition the clinical practice conducted by dentists, different microbiological and biochemical methods such as culturing, DNA probing and polymerase chain reaction (PCR) are employed to detect bacteria in samples from periodontal pockets. A general drawback of methods is that they do not quantify the severity of the disease.
So far, several studies aimed to develop new rapid point-of-care devices for the detection of Periodontal diseases, using markers that pinpoint the severity of Periodontitis. For example, Ivnitski et al, (2003) have used a hand-held non-invasive electrochemical amperometric device to detect salivary peroxidase, which is related to periodontitis. However, this method is nonspecific, expensive and time consuming.
Another example is the benzoyl-DL-arginine-naphthylamide (BANA) test strips that were developed by Loesche et al, (1990, 1992) and are currently in the market. This strip detects various bacteria (Treponema denticola, Porphyromonas gingivalis and Bacteroides forsythus) found in adult periodontal plaque. Even though, this method has been in the market for a while, but the strip only detects perio-pathogens and lack sensitivity and specificity.
There were a few other studies conducted to detect periodontitis using bacterial protease activity such as P. gingivalis as a marker. For instance earlier highly bacteria-specific fluorescence resonance energy transfer (FRET) peptides were generated to detect the proteolytic activity of P. gingivalis in vitro and in situ. The value of these substrates are beyond doubt, though, as protease activity in cases of periodontitis is a mixture of both host and microbiological origin these substrates only cover one part of the palet.
In the human oral cavity, there are around 700 microbial species. Previous studies have implicated Gram-negative anaerobic bacteria (called perio-pathogens) as the causative agents of periodontal diseases. A specific bacterial group, the “red complex” group which includes Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola, have been strongly associated with the clinical signs of advanced periodontal lesions. The current understanding of the pathogenesis of periodontitis suggests that the modulation of host response by bacterial products such as lipopolysaccharide and proteases are important factors in disease onset and progression.
As an effective host feedback response to bacterial challenge, neutrophils influx increases into gingival crevice. Neutrophils play a destructive role in the process of periodontal tissue breakdown due to the production of high levels of Human Neutrophil Elastase (HNE). This enzyme is a serine protease, which degrades elastin and other functionally and structurally important proteins in the periodontium during the inflammatory process. During which increased levels are found in the gingival crevicular fluid (GCF). In GCF the metabolic products of neutrophils, like HNE, are elevated and associated with periodontal inflammation. These proteins can thus be used as potential indicator for inflammation severity at individual sites. Notably, it is not only the amounts of this protease that differ, but also, the activity of the protease. For example, more elastase remains active in GCF during periodontal disease.
Another host-cell derived serine protease with potential for Periodontitis diagnosis is Cathepsin-G. In response to periodontitis this protease is secreted in the extracellular spaces where they degrade gingival tissue components such as collagen. Furthermore, there is an indication that Cathepsin-G is linked with the progression of gingivitis and chronic adult periodontitis. Immuno-histochemical studies of Cathepsin-G have shown that the protease is expressed and localized in the inflamed gingiva in an increased activity along with the severity of periodontal inflammation. These results suggested the possible involvement of Cathepsin-G in the degradation of inflamed gingival connective tissue since it was reported that Cathepsin-G level was elevated in the GCF.
To date, several methods have been described for elastase detection. These include chromatography, sephadex-gel-electrophoresis, and radioimmunoassay. Similarly, there are several studies which focus on the degradation of hydrogel films cross-linked with peptides such as HNE, Cathepsin-G and matrix metalloproteinase-8 (MMP-8), monitored using a combination of quartz crystal microbalance (QCM) and electrochemical impedance measurements. However even though these methods were recently introduced, there is some drawback by being complex and time consuming. There is need for a rapid point-of-care diagnostic detection method that would be of major benefit in the diagnosis, evaluation and treatment of disease severity.